Download Molecular tools for forensic body fluid identification

Molecular tools for forensic
body fluid identification
Hwan Young Lee, Ph.D.
Department of Forensic Medicine, Yonsei University College of
Medicine, Seoul, Korea
Presentation overview
 Introduction: Body fluid identification in forensics
 Recent developments in molecular genetics-based
genetics based body fluid
identification
- Messenger RNA profiling
- Bacterial ribosomal RNA analysis
- Micro RNA profiling
 Potential application of DNA methylation profiling for forensic body
fluid identification
1
Body fluid identification in forensics
 Information that supports a link between sample donors who have been
identified by DNA profiling and actual criminal acts is crucial.
 Body fluid identification, determining the sample’s cellular origin can reveal
significant insights into crime scene reconstruction and contribute toward
solving crimes.
Sex offender’s trial
Body fluids founds on the bed
Body fluid identification in forensics
 Current methods based on chemo luminescence or immunological tests are
mainly presumptive.
 Recent advances in genetics and molecular biology led to the suggestion of
the
use
of
a
molecular
genetics-based
approach
to
supplant
conventional methods.
Photo of belt with semen illuminated with white
light and UV light
Photo of robber mask with saliva illuminated with
white light and UV light
2
Recent developments
de elopments in molec
molecular
lar
genetics-based body fluid identification
RNA profiling for body fluid identification
 Some mRNAs and miRNAs are expressed in a tissue-specific manner and
their expression patterns can confirm specific body fluids, even after long
periods.
RNA Extraction –Unknown Body Fluid Sample
RNA Quantification
Detection of a specific transcript
present in sufficient quantity
cDNA Synthesis
(Reverse Transcription of Total RNA)
Detection of relative gene expression in
comparison to an endogenous control
Endpoint PCR
Quantitative RT PCR
Electropherogram Analysis
CT Value Analysis
Body Fluid Identification
Schema for body fluid identification using RNA profiling
3
mRNA profiling for body fluid identification
 Several mRNA-based multiplex PCR assays now are available for parallel
determination of venous blood, saliva, semen, and menstrual blood.
 Major advantages of mRNA profiling are the possibility of detecting several
body fluids in one multiplex reaction and the simultaneous DNA isolation
without loss of material.
Body fluid specificity of the mRNA markers
Multiplex result of body fluid mixture (Haas et al. FSIG 2009)
Microbial RNA for vaginal fluid identification
 The 16S-23S rRNA intergenic spacer region of Lactobacillus crispatus and
Lactobacillus gasseri were found to be a suitable marker for identifying
vaginal secretions, and could be incorporated into a mRNA multiplex system.
Multiplex result from a vaginal swab
(Fleming et al. FSIG 2010)
Multiplex result from a vaginal swab with semen
(TGM4: seminal fluid specific mRNA marker)
4
miRNA profiling for body fluid identification
 A clear advantage of microRNAs (miRNAs, 18-25 bases in length) over mRNA
is their small size, which makes them likely to have higher in vitro stability
than mRNAs.
 Accurate
quantification
of
miRNAs
using
quantitative RT PCR requires not only a highly
sensitive and specific detection platform for
experiment operation, but also a reproducible
methodology with an adequate model for data
analysis.
Differentiation of forensically relevant body fluids using genome-wide
miRNA expression data (Zubakov et al. IJLM 2010)
Potential
P
i l application
li i off DNA methylation
h l i
profiling for body fluid identification
5
DNA methylation
 DNA methylation is the addition of a methyl group to the DNA base
cytosine followed by a guanine (5' CG 3').
Cytosine
5-Methyl Cytosine
 DNA methylation of a gene's CpG island represses gene expression.
Different cell types have different methylation patterns, which
contributes to the differences in gene expression in different cell types.
tDMRs and body fluid identification
 Chromosome
pieces
called
tDMRs
(tissue-specific
differentially
methylated regions) show different DNA methylation profiles according to
the type of cell or tissue.
 The potential of tissue-specific differential DNA methylation for body
fluid identification was examined.
Table 1. Genomic information for candidate tDMRs for body fluid identification
Tissue
UCSC location (Mar. 2006)
CGI
Gene
Function
References
Testis
chr14:58182690–58182995
cpgi50
DACT1
Dapper 1 isoform 2
Genomics. 89:326
Testis
chr6:41881884 41882111
chr6:41881884–41882111
cpgi46
USP49
Ubiquitin carboxyl-terminal
carboxyl terminal hydrolase 49
Genomics 89:326
Genomics.
Blood
chr7:27135995–27136879
cpgi87
HOX44
Homeobox protein Hox-A4
PLoS Biol. 6:e22
Blood
chr5:176758438–176760564
cpgi82
PFN3
Profilin-3
PLoS Biol. 6:e22
Blood
chr21:46905647–46905874
cpgi55
PRMT2
Protein arginine N-methyltransferase 2
PLoS Biol. 6:e22
Lee et al. IJLM in press
6
Analysis of DNA methylation
 Sodium bisulfite treatment of CpG motifs
Met
CpG
p
Sodium bisulfite
CpG
Met
CpG
p
PCR
CpG
p
UpG
TpG
Sequencing
SBE
MSP
 Methylation-sensitive restriction enzyme treatment of CpG motifs in the
enzyme recognition sites
Met
CpG
Methylation-sensitive
restriction enzyme
CpG
PCR product
Met
CpG
PCR
CpG
No PCR product
Differential DNA methylation in body fluids
 DNA methylation profiles for the tDMRs for the genes DACT1, USP49,
HOXA4, PRMT2, and PFN3 were produced by sequencing of bisulfitetreated pooled DNA from blood, saliva, semen, menstrual blood, and
vaginal fluid obtained from 10 males and 6 females.
: DACT1
: USP49
7
Differential DNA methylation in body fluids
 DNA methylation profiles for the tDMRs for the genes DACT1, USP49,
HOXA4, PRMT2, and PFN3 were produced by sequencing of bisulfitetreated pooled DNA from blood, saliva, semen, menstrual blood, and
vaginal fluid obtained from 10 males and 6 females.
: HOXA4
: PRMT2
Differential DNA methylation in body fluids
 DNA methylation profiles for the tDMRs for the genes DACT1, USP49,
HOXA4, PRMT2, and PFN3 were produced by sequencing of bisulfitetreated pooled DNA from blood, saliva, semen, menstrual blood, and
vaginal fluid obtained from 10 males and 6 females.
: PFN3
8
DNA methylation and aging
 DNA methylation profiles of 3 tDMRs for the genes DACT1, PRMT2, and
USP49 were further analyzed by sequencing of bisulfite-treated pooled
DNA from blood, saliva, and semen obtained from 20 young (< 30 y) and
15 old (> 50 y) men.
men
a. Chr14:58182690–58182995 : DACT1
b. Chr21:46905841–46906149 : PRMT2
Y-BL
Y-SA
Y-SE
Y-BL
Y-SA
Y-SE
O-BL
O-SA
O-SE
O-BL
O-SA
O-SE
c. Chr6: 41881884–41882111 : USP49
Y-BL
Y-SA
Y-SE
O-BL
O-SA
O-SE
A multiplex PCR using methylationsensitive restriction enzyme
 A multiplex PCR was developed for determination of DNA methylation
status of 4 CpG loci at the USP49, DACT1, PRMT2, and PFN3 tDMRs
using HhaI,
HhaI which recognizes and cuts unmethylated GCGC sequence.
sequence
 Complete enzyme digestion was confirmed by the removal of PCR
product for the ACVR CpG island. DMSO was added at the amplification
step due to the high GC contents of the selected tDMRs (> 60%).
Amelogenin
USP49 DACT1 PRMT2 PFN3
DACT1
Amelogenin
USP49
PRMT2
D3S1358
ACVR
PCR without HhaI digestion
D3S1358
PFN3
ACVR
PCR after HhaI digestion
9
A multiplex PCR using methylationsensitive restriction enzyme
 The tDMRs for USP49, DACT1,
PRMT2, and PFN3 showed
semen-specific
ifi unmethylation.
th l ti
USP49 DACT1
Amelogenin
PFN3
PRMT2
Blood
 The tDMR for PFN3 showed
hypomethylation in menstrual
blood and vaginal fluid.
Saliva
Semen
Methylated control DNA
Amelogenin
USP49
DACT1 PRMT2 PFN3
Menstrual
blood
Unmethylated control DNA
Amelogenin
Vaginal fluid
A multiplex SBE for bisulfite-treated DNA
 A multiplex SBE was developed for determination of DNA methylation
status of 4 CpG loci at the USP49, DACT1, PRMT2, and PFN3 tDMRs.
 A multiplex SBE results were consistent with those of the multiplex PCR
using methylation sensitive restriction enzyme.
enzyme
PFN3
USP49
DACT1
USP49
PRMT2
DACT1
PRMT2
PFN3
Blood
Saliva
USP49
DACT1
PFN3
PRMT2
Totally unmethylated DNA profile
Semen
PFN3
Menstrual
blood
USP49
DACT1
PRMT2
DACT1
USP49
PFN3
PRMT2
Vaginal fluid
10
DNA methylation profiling for body fluid
identification
 The analysis of tissue-specific differential DNA methylation was
proposed
p
p
as a p
promising
g new method for the identification of forensic
body fluids.
 The multiplex PCR system, which allows combined use of tDMRs for
USP49, DACT1, PRMT2, and PFN3, could be used to discriminate
blood-saliva, semen and vaginal fluid-menstrual blood.
 Future genome-wide DNA methylation analysis using various body fluid
samples will be useful to identify additional body fluid-specific
fluid specific tDMRs and
enable the subsequent development of efficient analysis methods for
forensic casework.
Concluding remarks
 Body fluid identification can reveal significant insights into crime
scene reconstruction and contribute toward solving crimes.
 Along
Al
with
ith recentt advances
d
i genetics
in
ti and
d molecular
l
l biology,
bi l
l t
lots
of studies suggested RNA markers for differentiating body fluids,
but some of the results for miRNA markers have yet to be confirmed
and warrant additional investigation.
 The analysis of tissue-specific differential DNA methylation was
proposed
p
as a p
promising
g new method for the identification of
also p
body fluids, but additional marker development and forensic
validation will be necessary for practical use of this new approach in
forensic practices.
11
Acknowledgment
Eun Young Lee
Prof. Kyoung-Jin Shin
Myung Jin Park
Ja Hyun An
Dr. In Seok Yang
Ajin Choi
Eun Hye Kim
12